Synthetic mammalian signaling circuits for robust cell population control

Abstract

Disclosed herein include circuits, compositions, nucleic acids, populations, systems, and methods enabling cells to sense, control, and/or respond to their own population size. In some embodiments, an orthogonal communication channel allows specific communication between engineered cells. Also described herein, in some embodiments, is an evolutionarily robust ‘paradoxical’ regulatory circuit architecture in which orthogonal signals both stimulate and inhibit net cell growth at different signal concentrations. In some embodiments, engineered cells autonomously reach designed densities and/or activate therapeutic or safety programs at specific density thresholds. Methods of treatment are also provided in some embodiments.

1 . A nucleic acid composition, comprising:
 a first polynucleotide encoding a first synthase, wherein the first synthase is capable of catalyzing the synthesis of an orthogonal signal from a first precursor molecule,   a second polynucleotide encoding a second synthase, wherein the second synthase is capable of catalyzing the synthesis of the first precursor molecule from a second precursor molecule; and/or   a third polynucleotide encoding a transporter capable of transporting the orthogonal signal, the first precursor molecule, and/or the second precursor molecule across a cell membrane;   wherein the first polynucleotide, the second polynucleotide, and the third polynucleotide are operably linked to a ubiquitous mammalian promoter.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The nucleic acid composition of  claim 1 , comprising:
 a fourth polynucleotide encoding a signal-binding protein; and   a seventh polynucleotide encoding a third fusion protein comprising a signal-responsive domain and target protein,   wherein the signal-binding protein is capable of binding the orthogonal signal, wherein the signal-binding protein bound to the orthogonal signal is capable of reducing the stability, localization, and/or activity of the protein comprising the signal-responsive domain.   
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . The nucleic acid composition of  claim 6 , comprising:
 an eighth polynucleotide encoding one or more payloads, wherein the target protein is capable of modulating the concentration, localization, stability, and/or activity of the one or more payloads.   
     
     
         11 . (canceled) 
     
     
         12 . The nucleic acid composition of  claim 10 , wherein the target protein is capable of repressing the transcription of the one or more payloads; or
 wherein a payload transcript is capable of being translated to generate a payload protein, and wherein the target protein is capable of reducing the concentration, localization, stability, and/or activity of the payload protein.   
     
     
         13 .- 21 . (canceled) 
     
     
         22 . The nucleic acid composition of  claim 12 , wherein one or more of the payloads comprise a recombinase. 
     
     
         23 . The nucleic acid composition of  claim 22 , comprising:
 a second promoter and a ninth polynucleotide comprising a payload gene,
 wherein, in the absence of a recombination event, the second promoter and 
   the ninth polynucleotide are not operably linked,
 wherein the recombinase is capable of catalyzing the recombination event, and 
 wherein the second promoter and the ninth polynucleotide are operably linked after the recombination event such that the second promoter is capable of inducing transcription of the payload gene to generate a payload transcript. 
   
     
     
         24 . The nucleic acid composition of  claim 23 , wherein the recombination event comprises removal of a sequence flanked by recombinase target sites or an inversion of a sequence flanked by recombinase target sites; and wherein the ninth polynucleotide is flanked by recombinase target sites. 
     
     
         25 . (canceled) 
     
     
         26 . The nucleic acid composition of  claim 23 , wherein, prior to the recombination event, the sequence of the payload gene is inverted relative to the second promoter. 
     
     
         27 . The nucleic acid composition of  claim 23 , comprising at least one stop cassette situated between the second promoter and the payload gene, wherein the stop cassette comprises one or more stop sequences, and wherein the one or more stop cassettes are flanked by recombinase target sites, and wherein the at least one stop cassette is configured to prevent transcription of the payload gene and/or translation of the payload transcript. 
     
     
         28 . The nucleic acid composition of  claim 23 , wherein the payload transcript is capable of being translated to generate a payload protein. 
     
     
         29 . (canceled) 
     
     
         30 . The nucleic acid composition of  claim 1 , wherein the first synthase comprises an indole-3-acetic acid hydrolase, optionally iaaH, aux2 and/or AMI1, further optionally  A. tumefaciens  iaaH, eptienal % the second synthase comprises tryptophan 2-monooxygenase (iaaM), and the transporter is auxin exporter PIN2. 
     
     
         31 . (canceled) 
     
     
         32 . The nucleic acid composition of  claim 1 , wherein the first precursor molecule and/or the second precursor molecule is an endogenous molecule of a cell, optionally L-tryptophan. 
     
     
         33 . The nucleic acid composition of  claim 1 , wherein the orthogonal signal comprises indole-3-acetic acid (IAA), 1-napthalenatic acid (NAA), derivatives thereof, or any combination thereof, optionally the first precursor molecule comprises indole-3-acetamide (IAM) and/or 1-naphthaleneacetamide (NAM). 
     
     
         34 . The nucleic acid composition of  claim 6 , wherein the signal-binding protein comprises TIR1, and
 wherein the signal-responsive domain comprises an auxin inducible degron (AID), optionally a minimal auxin inducible degron (mAID).   
     
     
         35 . (canceled) 
     
     
         36 . The nucleic acid composition of  claim 1 , wherein the orthogonal signal: (i) exhibits minimal immunogenicity in a subject; (ii) is capable of diffusing through a target site of a subject, optionally the target site is a tissue of interest; and/or (iii) is orthogonal to the intrinsic signaling pathways of a subject. 
     
     
         37 . The nucleic acid composition of  claim 1 , wherein the orthogonal signal is a hormone, optionally auxin, abscisic acid or gibberellin. 
     
     
         38 .- 54 . (canceled) 
     
     
         55 . The nucleic acid composition of  claim 1 , wherein the expression and/or activity of the first synthase, second synthase, and/or transporter is configured to be responsive to changes in:
 cell localization;   one or more signal transduction pathways regulating cell survival, cell growth, cell proliferation, cell adhesion, cell migration, cell metabolism, cell morphology, cell differentiation, apoptosis, or any combination thereof, and/or   T cell activity, optionally T cell activity comprises one or more of T cell simulation, T cell activation, cytokine secretion, T cell survival, T cell proliferation, CTL activity, T cell degranulation, and T cell differentiation.   
     
     
         56 . The nucleic acid composition of  claim 1 , wherein a synthetic protein circuit component modulates the expression and/or activity of the first synthase, second synthase, and/or transporter, and/or wherein the expression and/or activity of the first synthase, second synthase, and/or transporter is configured to be responsive to immune cell stimulation, optionally immune cell stimulation comprises signal transduction induced by binding of a stimulatory molecule with its cognate ligand on the surface of an immune cell, optionally the cognate ligand is a CAR or a TCR. 
     
     
         57 . (canceled) 
     
     
         58 . (canceled) 
     
     
         59 . The nucleic acid composition of  claim 1 , wherein the nucleic acid composition is comprised within one or more vectors; optionally at least one of the one or more vectors is a viral vector, a plasmid, a naked DNA vector, a lipid nanoparticle, or any combination thereof, and further optionally the viral vector is an AAV vector, a lentivirus vector, a retrovirus vector, an integration-deficient lentivirus (IDLV) vector. 
     
     
         60 .- 99 . (canceled) 
     
     
         100 . The nucleic acid composition of  claim 22 , wherein the recombinase is Cre, Dre, Flp, KD, B2, B3, λ, HK022, HP1, γ6, ParA, Tn3, Gin, ΦC31, Bxb1, R4, derivatives thereof, or any combination thereof.